The proposed studies will determine the feasibility of compacting plasmid DNA into "nanoparticles" and using these nanoparticles to deliver their payload into cells of the central nervous system (CNS) as a non-viral, gene therapy technique. DNA compacting techniques will be used to form nanoparticles containing condensed DNA plasmids with diameters in the range of 8-12 nanometers. Preliminary data shows that non-targeted DNA nanoparticles (DNPs) can be injected directly into brain and produce long-term transgene expression brain cells, primarily in astrocytes. The principle studies will focus on synthesizing DNPs that can be administered intracerebrally or systemically and increase the transfection efficiency in neurons. Recent studies have demonstrated that bioconjugated quantum rods can be targeted to the transferrin receptor (TfR) and traverse the blood-brain barrier (BBB). These particles have an average size and shape that are similar to our DNPs. As these are key determinants in particle transport, it is reasonable to postulate that DNPs modified to target the TfR may cross the BBB. In preliminary studies we have succeeded in targeting the DNPs to neurons of the hippocampus in brain slices utilizing a ligand to the serpin enzyme complex receptor (sec-R), C105Y, which has been shown to be a novel cell-penetrating peptide;and its receptor, sec-R, has been identified on neurons. Taken together, we hypothesize that unimodal targeting of DNPs to the TfR will enable them to cross the BBB and non-specifically transfect neural cells (neurons and/or glia), while bimodal targeting of DNPs to the TfR and sec-R will enable them to cross the BBB and predominantly transfect neuronal cells. To achieve dopamine neuron specificity, we will use plasmid constructs that contain the tyrosine hydroxylase (TH) promoter;TH is the rate limiting enzyme in the biosynthetic pathway for dopamine. The study design in this two year project will focus on 1) the synthesis of targeted DNPs, 2) transfection efficiency of targeted DNPs in primary neuronal cultures or cell lines, 3) in vivo tracking and transfection efficiency of targeted DNPs using MRI, microPET, and bioluminescent imaging techniques as well as immunohistochemical and protein analyses, 4) toxicity of targeted DNPs in brain, and 5) feasibility of repeated administration of DNPs while maintaining safe and stable transgene expression in brain. Successful results in these studies could then be applied to animal models of neurodegenerative disorders and possibly lead to translational studies for the treatment of neurological disorders, such as Parkinson's disease. PUBLIC HEALTH RELEVANCE: The proposed studies will use a novel nanoparticle technology that allows nucleic acids (DNA) to be compacted near their theoretical limit;this technology almost duplicates the compaction efficiency of viruses. We present preliminary data showing proof-of-concept that these nanoparticles can be used as a non-viral gene therapy for transfecting cells in the brain. In the proposed studies, we will conjugate moieties to the nanoparticles that will target receptors at the blood-brain barrier (BBB) allowing them to cross from the brain vasculature into the brain tissue. Another conjugated moiety will specifically target a novel rapid uptake mechanism that has been identified on neurons in order to increase the neuronal transfection efficient of the nanoparticles. Successful results from our animal studies could then be translated to human studies using these targeted nanoparticles as a form of non-viral, gene therapy to treat various neurological disorders.